Nitrogen-fixing Soil Bacteria Research Articles

Effects of land application of municipal biosolids on nitrogen-fixing bacteria in agricultural soil

Effects of municipal biosolids on microbial N2-fixation in agricultural soil were assessed in a 3-month laboratory study which included analysis of metals, pharmaceuticals, and personal care products. Reference agricultural soil was amended with organic manure or municipal biosolids from a southern Ontario wastewater plant, with a biosolids-only treatment included to evaluate metabolic activity in this inoculum. Microbial N2-fixation in reference and manure-amended soils were similar (p = 0.144) over 3 months and lower than in biosolids-amended soil (p = 0.001); however, differences among soil treatments decreased over time, with no significant difference at test termination. In general, one-time application of biosolids caused short-term stimulation of N2-fixing activity with a return to reference conditions within 3 months. Although no residual effects were detected, biosolids introduced elevated levels of metals, pharmaceuticals, personal care products (particularly analgesics), and viable bacteria; long-term effects caused by repeat applications (as commonly practiced) requires further investigation.

Aerobic Vanillate Degradation and C 1 Compound Metabolism in Bradyrhizobium japonicum

Bradyrhizobium japonicum, a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome and is able to use low concentrations of vanillate, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of vanA1B, pcaG1H1, and genes for C(1) compound catabolism was upregulated in B. japonicum USDA110 cells grown in vanillate (N. Ito, M. Itakura, S. Eda, K. Saeki, H. Oomori, T. Yokoyama, T. Kaneko, S. Tabata, T. Ohwada, S. Tajima, T. Uchiumi, E. Masai, M. Tsuda, H. Mitsui, and K. Minamisawa, Microbes Environ. 21:240-250, 2006). To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption in vanA1B, pcaG1H1, and mxaF mutants of USDA110. The vanA1B and pcaG1H1 mutants were unable to grow in minimal media containing 1 mM vanillate and protocatechuate, respectively, although wild-type USDA110 was able to grow in both media, indicating that the upregulated copies of vanA1B and pcaG1H1 are exclusively responsible for vanillate degradation. Mutating mxaF eliminated expression of gfa and flhA, which contribute to glutathione-dependent C(1) metabolism. The mxaF mutant had markedly lower cell growth in medium containing vanillate than the wild-type strain. In the presence of protocatechuate, there was no difference in cell growth between the mxaF mutant and the wild-type strain. These results suggest that the C(1) pathway genes are required for efficient vanillate catabolism. In addition, wild-type USDA110 oxidized methanol, whereas the mxaF mutant did not, suggesting that the metabolic capability of the C(1) pathway in B. japonicum extends to methanol oxidation. The mxaF mutant showed normal nodulation and N(2) fixation phenotypes with soybeans, which was not similar to symbiotic phenotypes of methylotrophic rhizobia.

Plant–rhizobia mutualism influences aphid abundance on soybean

The mutualism between legumes and nitrogen-fixing soil bacteria (rhizobia) is a key feature of many ecological and agricultural systems, yet little is known about how this relationship affects aboveground interactions between plants and herbivores. We investigated the effects of the rhizobia mutualism on the abundance of a specialized legume herbivore on soybean plants. In a field experiment, soybean aphid (Aphis glycines) abundances were measured on plants (Glycine max) that were either (1) treated with a commercial rhizobial inoculant, (2) associating solely with naturally occurring rhizobia, or (3) given nitrogen fertilizer. Plants associating with naturally occurring rhizobia strains exhibited lower aphid population densities compared to those inoculated with a commercial rhizobial preparation or given nitrogen fertilizer. Genetic analyses of rhizobia isolates cultured from field plants revealed that the commercial rhizobia strains were phylogenetically distinct from naturally occurring strains. Plant size, leaf nitrogen concentration, and nodulation density were similar among rhizobia-associated treatments and did not explain the observed differences in aphid abundance. Our results demonstrate that plant–rhizobia interactions influence plant resistance to insect herbivores and that some rhizobia strains confer greater resistance to their mutualist partners than do others.

The Na+-translocating NADH : ubiquinone oxidoreductase of Azotobacter vinelandii negatively regulates alginate synthesis

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that produces the exopolysaccharide alginate. In this report we describe the isolation and characterization of A. vinelandii strain GG4, which carries an nqrE : : Tn5 mutation resulting in alginate overproduction. The nqrE gene encodes a subunit of the Na+-translocating NADH : ubiquinone oxidoreductase (Na+-NQR). As expected, Na+-NQR activity was abolished in mutant GG4. When this strain was complemented with the nqrEF genes this activity was restored and alginate production was reduced to wild-type levels. Na+-NQR may be the main sodium pump of A. vinelandii under the conditions tested ( approximately 2 mM Na+) since no Na+/H+-antiporter activity was detected. Collectively our results indicate that in A. vinelandii the lack of Na+-NQR activity caused the absence of a transmembrane Na+ gradient and an increase in alginate production.

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.

FlhDC, but not fleQ, regulates flagella biogenesis in Azotobacter vinelandii, and is under AlgU and CydR negative control

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Upon encystment, this bacterium becomes non-motile. As in enteric bacteria, motility in A. vinelandii occurs through the use of peritrichous flagella. Pseudomonas aeruginosa, a phylogenetically close relative of A. vinelandii, possesses a single polar flagellum. The FlhDC proteins are the master regulators of flagella and motility in enterobacteria, whereas FleQ is the master regulator in P. aeruginosa, and it is under AlgU (sigmaE) negative control. At present, nothing is known about the organization and expression of flagella genes in A. vinelandii. Here, we identified the flagella gene cluster of this bacterium. Homologues of the master regulatory genes flhDC and fleQ are present in A. vinelandii. Inactivation of flhDC, but not fleQ, impaired flagella biogenesis and motility. We present evidence indicating that a negative effect of the AlgU sigma factor on flhDC expression causes loss of motility in A. vinelandii, and that CydR (a homologue of Fnr) is under AlgU control and has a negative effect on flhDC expression. Taken together, these results suggest the existence of a cascade consisting of AlgU and CydR that negatively controls expression of flhDC; the results also suggest that the block in flagella synthesis under encystment conditions centres on flhDC repression by the AlgU–CydR cascade.

A Large Scale Analysis of Protein-Protein Interactions in the Nitrogen-fixing Bacterium Mesorhizobium loti

Global viewing of protein–protein interactions (PPIs) is a useful way to assign biological roles to large numbers of proteins predicted by complete genome sequence. Here, we systematically analyzed PPIs in the nitrogen-fixing soil bacterium Mesorhizobium loti using a modified high-throughput yeast two-hybrid system. The aims of this study are primarily on the providing functional clues to M. loti proteins that are relevant to symbiotic nitrogen fixation and conserved in other rhizobium species, especially proteins with regulatory functions and unannotated proteins. By the screening of 1542 genes as bait, 3121 independent interactions involving 1804 proteins (24% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure and the general features of the interacting partners. Most PPIs detected in this study are novel interactions revealing potential functional relationships between genes for symbiotic nitrogen fixation and signal transduction. Furthermore, we have predicted the putative functions of unannotated proteins through their interactions with known proteins. The results described here represent new insight into protein network of M. loti and provide useful experimental clues to elucidate the biological function of rhizobial genes that can not be assigned directly from their genomic sequence.

Differential Expression of the TFIIIA Regulatory Pathway in Response to Salt Stress between Medicago truncatula Genotypes

Soil salinity is one of the most significant abiotic stresses for crop plants, including legumes. These plants can establish root symbioses with nitrogen-fixing soil bacteria and are able to grow in nitrogen-poor soils. Medicago truncatula varieties show diverse adaptive responses to environmental conditions, such as saline soils. We have compared the differential root growth of two genotypes of M. truncatula (108-R and Jemalong A17) in response to salt stress. Jemalong A17 is more tolerant to salt stress than 108-R, regarding both root and nodulation responses independently of the nitrogen status of the media. A dedicated macroarray containing 384 genes linked to stress responses was used to compare root gene expression during salt stress in these genotypes. Several genes potentially associated with the contrasting cellular responses of these plants to salt stress were identified as expressed in the more tolerant genotype even in the absence of stress. Among them, a homolog of the abiotic stress-related COLD-REGULATEDA1 gene and a TFIIIA-related transcription factor (TF), MtZpt2-1, known to regulate the former gene. Two MtZpt2 TFs (MtZpt2-1 and MtZpt2-2) were found in Jemalong A17 plants and showed increased expression in roots when compared to 108-R. Overexpression of these TFs in the sensitive genotype 108-R, but not in Jemalong A17, led to increased root growth under salt stress, suggesting a role for this pathway in the adaptive response to salt stress of these M. truncatula genotypes.

Genetic Determinants of Swarming in Rhizobium etli

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.

Complexation of oxoanions and cationic metals by the biscatecholate siderophore azotochelin

Azotochelin is a biscatecholate siderophore produced by the nitrogen-fixing soil bacterium Azotobacter vinelandii. The complexation properties of azotochelin with a series of oxoanions [Mo(VI), W(VI) and V(V)] and divalent cations [Cu(II), Zn(II), Co(II) and Mn(II)] were investigated by potentiometry, UV-vis and X-ray spectroscopy. Azotochelin forms a strong 1:1 complex with molybdate (log K=7.6+/-0.4) and with tungstate and vanadate; the stability of the complexes increases in the order Mo<V<W (log KappMo=7.3+/-0.4; log KappV=8.8+/-0.4 and log KappW=9.0+/-0.4 at pH 6.6). The Mo atom in the 1:1 Mo-azotochelin complex is bound to two oxo groups in a cis position and to the two catecholate groups of azotochelin, resulting in a slightly distorted octahedral configuration. Below pH 5, azotochelin appears to form polynuclear complexes with Mo in addition to the 1:1 complex. Azotochelin also forms strong complexes with divalent metals. Of the metals studied, Cu(II) binds most strongly to azotochelin (log betaCuLH2-=-12.9+/-0.1), followed by Zn(II) log betaZnL3-=-24.1+/-0.14, log betaZnLH2-=-17.83+/-0.09), Mn(II) (log betaMnL3-=-29, log betaMnLH2-=-18.6+/-0.8, log betaMnLH2-=-11.5+/-0.7) and Co(II) (log betaCoLH2-=-23.0+/-0.3, log betaCoLH2-=-13.5+/-0.2). Since very few organic ligands are known to bind strongly to oxoanions (and particularly molybdate) at circumneutral pH, the unusual properties of azotochelin may be used for the separation and concentration of oxoanions in the laboratory and in the field. In addition, azotochelin may prove useful for the investigation of the biogeochemistry of Mo, W and V in aquatic and terrestrial systems.

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL−1) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.

Cloning, Sequencing, and Characterization of the Azospirillum brasilense fhuE Gene

The fhuE gene of Escherichia coli encodes the FhuE protein, which is a receptor protein in the coprogen-mediated siderophore iron-transport system. A fhuE gene homologue from Azospirillum brasilense, a nitrogen-fixing soil bacterium that lives in association with the roots of cereal grasses, was cloned, sequenced, and characterized. The A. brasilense fhuE encodes a protein of 802 amino acids with a predicted molecular weight of approximately 87 kDa. The deduced amino-acid sequence showed a high level of homology to the sequences of all the known fhuE gene products. The fhuE mutant was sensitive to iron starvation and defective in coprogen-mediated iron uptake. The mutant failed to express one membrane protein of approximately 78 kDa that was induced by iron starvation in the wild type. Complementation studies showed that the A. brasilense fhuE gene, when present on a low-copy number plasmid, could restore the functions of the mutant. Mutation in fhuE gene did not affect nitrogen fixation.

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2',3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.

Increase in the Nitrogen Content of Soil by the Introduction of Earthworms into Soil

The contribution of an earthworm species (Amynthas vittatus) to the increase of the nitrogen content of soil was examined. Three specimens of adult earthworms were introduced into 300 g of soil (Gray Lowland soil, silty clay) supplemented with 1% carboxymethyl cellulose in a container and incubated for 32 d at 22°C in the dark. The contents of total-N, NH4-N and NO3-N, and the population of aerobic nitrogen-fixing bacteria in soil significantly increased after incubation with the earthworms, while the natural abundance of 15N (δ15N) in soil decreased. The amount of nitrogen in the earthworms did not decrease during the incubation in the microcosm. Both acetylene reduction activity of the microcosm and incorporation of 15N to soil from atmospheric 15N2 were significantly enhanced by the introduction of the earthworms into soil, though the observed increment of nitrogen in soil was much higher than the estimated one based on the nitrogen-fixing activity. The results obtained in the present study indicated that the earthworms increased the nitrogen content of soil, presumably due to the enhancement of the nitrogen-fixing activity of the soil from the microcosm by the earthworms.

5-n-Alkylresorcinols from the Nitrogen-fixing Soil Bacterium Azotobacter chroococcum Az12

A mixture of five saturated 5-n-alkylresorcinol homologues was isolated from vegetative cells of the nitrogen-fixing soil bacterium Azotobacter chroococcum Az12. Their structures were established by spectrometry (1H NMR, EI-MS, FAB-MS, FAB-MS/MS) and chromatography (GC, TLC) means.

Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes.

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.

Bacterial iron transport: coordination properties of azotobactin, the highly fluorescent siderophore of Azotobacter vinelandii.

Azotobacter vinelandii, a nitrogen-fixing soil bacterium, secretes in iron deficiency azotobactin delta, a highly fluorescent pyoverdin-like chromopeptidic hexadentate siderophore. The chromophore, derived from 2,3-diamino-6,7 dihydroxyquinoline, is bound to a peptide chain of 10 amino acids: (L)-Asp-(D)-Ser-(L)-Hse-Gly-(D)-beta-threo-HOAsp-(L)-Ser-(D)-Cit-(L)-Hse-(L)-Hse lactone-(D)-N(delta)-Acetyl, N(delta)-HOOrn. Azotobactin delta has three different iron(III) binding sites which are one hydroxamate group at the C-terminal end of the peptidic chain (N(delta)-Acetyl, N(delta)-HOOrn), one alpha-hydroxycarboxylic function in the middle of the chain (beta-threo-hydroxyaspartic acid), and one catechol group on the chromophore. The coordination properties of its iron(III) and iron(II) complexes were measured by spectrophotometry, potentiometry, and voltammetry after the determination of the acid-base functions of the uncomplexed free siderophore. Strongly negatively charged ferric species were observed at neutral p[H]'s corresponding to a predominant absolute configuration Lambda of the ferric complex in solution as deduced from CD measurements. The presence of an alpha-hydroxycarboxylic chelating group does not decrease the stability of the iron(III) complex when compared to the main trishydroxamate siderophores or to pyoverdins. The value of the redox potential of ferric azotobactin is highly consistent with a reductive step by physiological reductants for the iron release. Formation and dissociation kinetics of the azotobactin delta ferric complex point out that both ends of this long siderophore chain get coordinated to Fe(III) before the middle. The most striking result provided by fluorescence measurements is the lasting quenching of the fluorophore in the course of the protonation of the ferric azotobactin delta complex. Despite the release of the hydroxyacid and of the catechol, the fluorescence remains indeed quenched, when iron(III) is bound only to the hydroxamic acid, suggesting a folded conformation at this stage, around the metal ion, in contrast to the unfolded species observed for other siderophores such as ferrioxamine or pyoverdin PaA.

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO 2) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.

The bactericidal activity of lectins from nitrogen-fixing bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.

Biological role of lectins from Bacillus polymyxa during interaction with surface carbohydrates of wheat roots

The data obtained demonstrate the capacity of lectins LI and LII from the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 to change their proteolytic activity after interaction with surface carbohydrates of wheat seedling roots.